Electrophoresis apparatus and method involving parallel channels

ABSTRACT

An electrophoresis apparatus includes a substrate (34) which supports a filling region (22) and a plurality of electrophoresis lanes (20). The filling region (22) communicates a sample to the plurality of electrophoresis lanes (20). A method of electrophoresis includes providing the above-described electrophoresis apparatus, applying a sample to the filling region (22), the plurality of electrophoresis lanes (20) receiving the sample from the filling region (22), and electrophoresing the sample in the plurality of electrophoresis lanes (20).

RELATED APPLICATION

The present application is related to the following applications:

"Methods and Systems for Biological Reagent Placement", having Ser. No.08/648,635, filed on May 13, 1996, now U.S. Pat. No. 5,731,152;

"Automated Electrophoresis System and Method", having Ser. No.08/788,970, MNE00503 filed on Jan. 24, 1997, now U.S. Pat. No.5,851,370; and

"Assay Dispensing Apparatus", having Ser. No. 08/789,220, MNE00502 filedon Jan. 24, 1997, now U.S. Pat. No. 5,772,966.

The subject matter of the above-identified related applications ishereby incorporated by reference into the disclosure of thisapplication.

FIELD OF THE INVENTION

The present invention relates to electrophoresis devices and methods.

BACKGROUND OF THE INVENTION

Gel electrophoresis devices commonly include a sheet or slab of gelmaterial sandwiched between two glass plates. A comb is utilized to forma plurality of sample wells in the gel. Each sample well corresponds toa single electrophoresis lane.

A respective sample is loaded into each sample well. Thereafter, avoltage is applied across the gel to generate an electric field tosimultaneously electrophorese all of the samples. As the samples migratein the gel due to the electric field, there is a potential forinteraction or bleeding between adjacent lanes that are closelypositioned. The potential for interaction limits a number of physicallanes which can be provided using this approach.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims.However, other aspects of the present invention are disclosed in thefollowing detailed description and the accompanying drawings in which:

FIG. 1 is a block diagram of a first embodiment of an electrophoresisdevice in accordance with the present invention;

FIG. 2 is a perspective view of a second embodiment of anelectrophoresis device in accordance with the present invention;

FIG. 3 is a sectional view of the first plate illustrated in FIG. 2;

FIG. 4 is a cross-sectional view of a first embodiment of an apparatusfor loading a sample into a third embodiment of an electrophoresisdevice;

FIG. 5 is a perspective view of a fourth embodiment of anelectrophoresis device in accordance with the present invention;

FIG. 6 is a perspective view that illustrates a second embodiment of anapparatus for applying samples to an electrophoresis device;

FIG. 7 is a general diagram that illustrates a third embodiment of anapparatus for applying samples and a buffer solution to anelectrophoresis device;

FIG. 8 is a general diagram that illustrates an embodiment of anelectrophoresis system in accordance with the present invention;

FIG. 9 is a flow chart of an embodiment of a method of electrophoresisin accordance with the present invention;

FIG. 10 illustrates an example plot of a measured quantity versus aprogression quantity;

FIG. 11 is a flow chart of an embodiment of a method of determining acharacteristic of the sample based on a collection of data;

FIG. 12 illustrates an example of a linear transformation fordetermining sizes of molecules in the sample;

FIG. 13 illustrates an example of a second transformation fordetermining sizes of molecules in the sample;

FIG. 14 is a general diagram that illustrates an embodiment of anapparatus for sensing sample molecules for use in embodiments of theelectrophoresis device;

FIG. 15 is a block diagram of an embodiment of an apparatus for applyinga plurality of samples to an electrophoresis device;

FIG. 16 is a perspective view of another embodiment of an apparatus forapplying a plurality of samples to an electrophoresis device;

FIG. 17 is an illustration of the embodiment of the apparatus of FIG. 16in a covered state;

FIG. 18 is a top view of a third embodiment of an apparatus fordispensing a plurality of samples to an electrophoresis device;

FIG. 19 is a block diagram of an embodiment of an automatedelectrophoresis system in accordance with the present invention;

FIG. 20 is a diagram that illustrates an embodiment of a transportmechanism in accordance with the present invention;

FIG. 21 illustrates another approach to contemporaneouslyelectrophoresing samples supported by a plurality of electrophoresisdevices;

FIG. 22 is a flow chart of an embodiment of a method of electrophoresisfor a single electrophoresis device; and

FIG. 23 is a block diagram of a second embodiment of an automatedelectrophoresis system in accordance with the present invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

FIG. 1 is a block diagram of a first embodiment of an electrophoresisdevice in accordance with the present invention. The electrophoresisdevice includes a first plurality of electrophoresis lanes 20 incommunication with a first filling region 22. The first filling region22 is utilized to receive a first sample of molecules for separation ineach of the first plurality of electrophoresis lanes 20. The firstsample can be separated in the first plurality of electrophoresis lanes20 using an electrophoresis process, such as a gel electrophoresisprocess or a capillary electrophoresis process which are known in theart.

It is noted that the term "sample" in the present application isinclusive of a variety of samples, including but not limited to medicalsamples, genomic samples, environmental samples, and agriculturalsamples. Of particular interest is a sample having molecular chains ofat least one nucleotide. Here, for example, the sample can include aprocessed DNA sample or a processed RNA sample from a plant or a livingorganism, such as a human. In these applications, the sample typicallyincludes a plurality of fragments of DNA or RNA formed using arestriction enzyme.

Optionally, the molecules in the sample are tagged with a member whichcan be sensed by a molecular sensor. Such members are commonly referredto in the art as tags, markers, reporters, and labels. Examples of suchmembers include, but are not limited to, radioactive members, opticalmembers (such as fluorescent members, luminescent members, andlight-scattering members), charged members, and magnetic members.

By electrophoresing the sample, one or more characteristics of moleculescontained in the sample can be determined. For example, the sample canbe electrophoresed to detect a predetermined molecular structure in thesample. The predetermined molecular structure can indicate, for example:(i) the presence of a pathogen in an environmental sample such as water;(ii) crop resistance in an agricultural sample; or (iii) a disease genein a medical sample. Alternatively, the sample can be electrophoresed todetermine a molecular structure associated with the sample. Here, forexample, the sample is electrophoresed to determine a nucleotide basesequence associated therewith. As another alternative, the sample can beelectrophoresed in a fragment sizing application.

The electrophoresis device can also include a second plurality ofelectrophoresis lanes 24 in communication with a second filling region26. The second filling region 26 is utilized to receive a second sampleof molecules for separation in each of the second plurality ofelectrophoresis lanes 24. The second sample can be separated in thesecond plurality of electrophoresis lanes 24 in the same manner as thefirst sample.

Preferably, the electrophoresis device provides a physical barrierbetween the combination of the first plurality of electrophoresis lanes20 and the first filling region 22, and the combination of the secondplurality of electrophoresis lanes 24 and the second filling region 26.As a result, communication of the first sample to the second pluralityof electrophoresis lanes 24 or to the second filling region 26 isinhibited. Similarly, communication of the second sample to the firstplurality of electrophoresis lanes 20 or to the first filling region 22is inhibited.

In general, the electrophoresis device can include any number of fillingregions rather than just the two described above. Further, each fillingregion can have any respective plurality of electrophoresis lanes incommunication therewith. Depending on the size of the device and thewidth of the lanes, the device can define hundreds of lanes, thousandsof lanes, tens of thousands of lanes, or hundreds of thousands of lanes.Regardless of the number of lanes, the electrophoresis device canprovide physical barriers, as described earlier, which inhibit crosscontamination of samples between predetermined pairs of lanes.

The electrophoresis device further comprises at least one molecularsensor 30 associated with the first plurality of electrophoresis lanes20 to sense molecules which pass by its location during electrophoresis.Examples of the at least one molecular sensor 30 include, but are notlimited to, an optical sensor, an electrical sensor, and a radioactivesensor. The at least one molecular sensor 30 may directly sense untaggedmolecules or indirectly sense molecules by sensing the tagging membersassociated therewith.

As is known in the art, the process of electrophoresis separates thesample of molecules in dependence upon the size of the molecules. Inmany applications, a number of bands of like-sized molecules are formedduring electrophoresis. In these applications, each of the at least onemolecular sensor 30 senses bands of like-sized molecules which passthereby.

Preferably, the at least one molecular sensor 30 includes at least onemolecular sensor for each of the first plurality of electrophoresislanes 20. If desired, the at least one molecular sensor 30 can include aplurality of molecular sensors distributed along each electrophoresislane. It is also preferred that each of the at least one molecularsensor 30 has a fixed position and/or location along its respectiveelectrophoresis lane.

The electrophoresis device further comprises at least one molecularsensor 32 associated with the second plurality of electrophoresis lanes24. Preferably, the at least one molecular sensor 32 includes at leastone molecular sensor for each of the second plurality of electrophoresislanes 24. If desired, the at least one molecular sensor 32 can include aplurality of molecular sensors distributed along each electrophoresislane. It is also preferred that each of the at least one molecularsensor 32 has a fixed position and/or location along its respectiveelectrophoresis lane.

Each of the at least one molecular sensor 32 is utilized to sensemolecules which pass by its location during electrophoresis. As with theat least one molecular sensor 30, each of the at least one molecularsensor 32 can be utilized to sense bands of like-sized molecules whichpass thereby.

The first plurality of electrophoresis lanes 20, the first fillingregion 22, the second plurality of electrophoresis lanes 24, and thesecond filling region are supported by a substrate 34. In a preferredembodiment, the substrate 34 is micropatterned to define the firstplurality of electrophoresis lanes 20, the first filling region 22, thesecond plurality of electrophoresis lanes 24, and the second fillingregion 26. Additionally, the at least one molecular sensor 30 and the atleast one molecular sensor 32 can be integrated with the substrate 34,or can be external to the substrate 34.

FIG. 2 is a perspective view of a second embodiment of anelectrophoresis device in accordance with the present invention. Theelectrophoresis device includes a first plate 40 and a second plate 42.The first plate 40 includes a substrate 44 patterned to define a firstplurality of channels or troughs, including channels 46 and 48, a secondplurality of channels including channels 50 and 52, a first fillingregion 54, and a second filling region 56. The second plate 42 isutilized to cover a face of the first plate 40 at which the channels 46,48, 50, and 52 and the filling regions 54 and 56 are defined.

The first plate 40 and the second plate 42 can be formed of materialsincluding, but not limited to, insulator materials, semiconductormaterials, glass, polymers, or plastic. The channels 46, 48, 50, and 52and the filling regions 54 and 56 can be formed during molding of thesubstrate 44 or can be etched or machined into the substrate 44.

A suitable gel, such as an acrylamide gel or an agarose gel, ispreferably cast into the channels 46, 48, 50, and 52. With the secondplate 42 removed from the first plate 40, the channels 46, 48, 50, and52 can be filled with the gel using a doctor blade or a similartechnique. Thereafter, the second plate 42 is secured over the firstplate 40 to enclose the gel within the channels 46, 48, 50, and 52.Alternatively, the channels 46, 48, 50, and 52 can be filled with thegel while the second plate 42 is secured over the first plate 40. Inthis case, the gel can be pumped through the channels 46, 48, 50, 52, orcan be drawn through the channels 46, 48, 50, and 52 using a vacuum.

With the second plate 42 covering the first plate 40, a first sample ofmolecules is applied to the first filling region 54 and a second sampleof molecules is applied to the second filling region 56. The firstfilling region 54 communicates the first sample to the first pluralityof channels, including the channels 46 and 48. The first plurality ofchannels provides a first plurality of electrophoresis lanes for thefirst sample. The second filling region 56 communicates the secondsample to the second plurality of channels, including the channels 50and 52. The second plurality of channels provides a second plurality ofelectrophoresis lanes for the second sample.

Associated with each channel is a pair of electrodes for generating anelectric field therein. With reference to the embodiment illustrated inFIG. 2, electrodes 60, 62, 64, and 66 are integrated with the substrate44 at a terminal end of the channels 46, 48, 50, and 52, respectively.Electrodes 70, 72, 74, and 76 are integrated with the substrate 44 neara sample-receiving end of the channels 46, 48, 50, and 52, respectively.As illustrated, portions of the electrodes 70 and 72 can be located atthe first filling region 54, and portions of the electrodes 74 and 76can be located at the second filling region 56. If desired, theelectrodes 70 and 72 can be merged as a single electrode, and theelectrodes 74 and 76 can be merged as a single electrode. The electrodes60, 62, 64, 66, 70, 72, 74, and 76 can be formed of a semiconductormaterial or a conductor material, such as a metal or a polymer,integrated with the substrate 44.

An electric field is generated in a channel by applying a voltage acrossits associated pair of electrodes. For example, an electric field isgenerated in the channel 46 by applying a voltage between the electrode70 and the electrode 60. The electric field is applied to electrophoresethe samples in the filling regions.

By including a respective pair of electrodes for each channel, theelectric field in each channel can be independently controlled. Thisconfiguration affords the flexibility of: (i) applying a common electricfield to all of the channels; (ii) applying a common electric field toall channels in communication with a common filling region, with thepossibility of applying a different electric field to channels incommunication with a different filling region; or (iii) applying adifferent electric field to different channels associated with a commonfilling region.

To sense molecular transport induced by the electric field, a pair ofsensing electrodes is associated with each channel. With reference tothe embodiment illustrated in FIG. 2, a first electrode 80 and a secondelectrode 82 are proximate to the channel 46, a first electrode 84 and asecond electrode 86 are proximate to the channel 48, a first electrode90 and a second electrode 92 are proximate to the channel 50, and afirst electrode 94 and a second electrode 96 are proximate to thechannel 52. Each first electrode and second electrode are located atopposite sides along a width of its associated channel. Preferably, theelectrodes 80, 82, 84, 86, 90, 92, 94, and 96 are formed of asemiconductor material or a conductor material, such as a metal or apolymer, integrated with the substrate 44.

If desired, each first electrode and second electrode can abut aninterior of the channel to provide contact with the gel and/or thesample therein. Alternatively, each channel can have a first insulatinglayer and a second insulating layer which electrically insulates thefirst electrode and the second electrode, respectively, from theinterior of the channel. The insulating layers can serve to capacitivelycouple the electrodes to the channel, and to protect the electrodes fromcorrosion.

Each pair of sensing electrodes performs an impedance measurement at apredetermined location in the channel. The impedance measurement is usedto determine the presence of molecules from the sample at the location.Either a real part or an imaginary part of the impedance can bemeasured. Typically, the impedance varies from a nominal level asmolecules from the sample become proximate to the sensing electrodes.The impedance can increase or decrease from the nominal level based uponthe charged state of the molecules.

Optionally, the first plate 40 includes a heat sink 97 which draws heatgenerated during electrophoresis from the first plurality of channelsand the second plurality of channels. Preferably, the heat sink 97 isintegrated with the substrate 44 at an opposite face relative to theface at which the channels are defined.

The heat sink 97 acts to reduce temperature differences both betweenchannels and within channels. By equalizing the temperature betweenchannels, the channel-to-channel variability of electrophoretictransport of like molecules is reduced. By equalizing the temperaturewithin a channel, substantially-linear bands of molecules are formedduring electrophoresis.

The heat sink 97 can be formed of a thermally-conductive material topassively draw heat from the channels. Alternatively, the heat sink 97can include a thermoelectric member which actively cools the channels inresponse to an electrical signal applied thereto.

As with the embodiment described with reference to FIG. 1, it is notedthat the electrophoresis device of FIG. 2 can generally include anynumber of filling regions, rather than the two described, and any numberof channels per filling region.

The second plate 42 can be a plate dedicated for covering the firstplate 40, or can serve other purposes. For example, the second plate 42can include one or more of the elements described for the first plate40. Here, the second plate 42 can be stacked on top of the first plate40 so that electrophoresis can be performed using both plates. In thisway, any plurality of like plates can be stacked to performelectrophoresis.

Another purpose which can be served by the second plate 42 is to assistin sensing molecular transport in the first plate 40. Here, in general,the second plate 42 can include one or more molecular sensors forsensing the molecular transport. In one embodiment, the second plate 42includes an electrode 98 which is located proximate to a channel whenthe second plate 42 covers the first plate 40. Molecular transport canbe sensed by sensing an impedance between the electrode 98 and anelectrode 99 associated with the first plate 40.

FIG. 3 is a sectional view of the first plate 40 illustrated in FIG. 2.As illustrated, a dimension 100 of the first filling region 54 narrowsfrom a sample-receiving opening 102 to openings 104 to the channels 46and 48. Similarly, a dimension 106 of the second filling region 56narrows from a sample-receiving opening 110 to openings 112 to thechannels 50 and 52.

In general, the channels 46, 48, 50, and 52 and the filling regions 54and 56 can be dimensioned and spaced as desired. Preferably, the fillingregions 54 and 56 are dimensioned to have a predetermined volume. Thepredetermined volume is selected in accordance with a predeterminedamount of sample which is to be applied to the channels.

In a preferred embodiment, each channel has a width 114 of 50micrometers or less, a height of 50 micrometers or less, and a length116, preferably between one and ten centimeters. A distance 118 betweenadjacent channels in communication with the same filling region isapproximately the same as the width 114 of each channel. A distance 119between adjacent channels in communication with different fillingregions is greater than the width 114 of each channel.

The filling regions 54 and 56 each have a width 120 dependent upon thenumber of channels which communicate therewith and the dimensions of thechannels. For two 50-micrometer channels per filling region, thecross-sectional width can be 250 micrometers or less. For three50-micrometer channels per filling region, the width 120 can be 400micrometers or less. In a preferred embodiment, the filling regions 54and 56 have a height of 50 micrometers or less.

In general, the dimensions of the channels and the filling regions areselected with consideration to a resulting diffusion time of themolecules through the channels, and a resulting sensitivity fordetecting bands of molecules using the electrodes 80, 82, 84, 86, 90,92, 94, and 96.

FIG. 4 is a cross-sectional view of a first embodiment of an apparatusfor loading a sample into a third embodiment of an electrophoresisdevice 130. The electrophoresis device 130 defines three filling regions132, 134, and 136, and nine channels 138, 140, 142, 144, 146, 148, 150,152, and 154, in a manner similar to the embodiment of FIGS. 2 and 3.The filling region 132 communicates a first sample received therein tothe channels 138, 140, and 142. The filling region 134 communicates asecond sample received therein to the channels 144, 146, and 148. Thefilling region 136 communicates a third sample received therein to thechannels 150, 152, and 154.

The apparatus includes a gasket 160 for loading a sample into a fillingregion, such as loading the second sample into the filling region 134. Agasket 160 provides a terminal member of a sample dispensing apparatussuch as a syringe or a sample handling robot. Preferably, the gasket 160is formed of an elastomeric material such as teflon or rubber.

The gasket 160 defines an opening 162 through which the second sample isloaded into the filling region 134. The opening 162 is dimensioned to besmaller than an opening 164 of the filling region 134. An outerperiphery 166 of the gasket 160 is dimensioned to fully surround theopening 164 of the filling region 134. When forced into contact with theelectrophoresis device 130, the gasket 160 seals the filling region 134from adjacent filling regions, namely the filling region 132 and thefilling region 136, and from an external environment. Once sealed, thesample can be injected into the filling region 134 by pressurization.When the filling region 134 becomes filled with the sample, the gasket160 is removed from the electrophoresis device.

The gasket 160 is advantageous in: (i) confining the sample to thefilling region of interest; (ii) inhibiting the sample fromcontaminating adjacent filling regions; and (iii) dispensing apredetermined volume of sample based upon the volume of the fillingregion of interest. In addition, by utilizing filling regions which aresignificantly wider (e.g. 400 micrometers) than the channels (e.g. 50micrometers), placement tolerances for the gasket 160 are relaxed andcross contamination of samples is reduced.

FIG. 5 is a perspective view of a fourth embodiment of anelectrophoresis device in accordance with the present invention. Theelectrophoresis device includes a first plate 170 and a second plate172. The first plate 170 includes a substrate 174 patterned to define aplurality of channels, including channels 176, 178, 180, and 182. Thesecond plate 172 is utilized to cover a face of the first plate 170 atwhich the plurality of channels are defined.

The first plate 170 and the second plate 172 can be formed of similarmaterials as the first plate 40 and the second plate 42 described withreference to FIG. 2. As with the embodiment of FIG. 2, the channels 176,178, 180, and 182 can be formed during molding of the substrate 174 orcan be etched or machined into the substrate 174.

A suitable gel is cast into the channels 176, 178, 180, and 182. Thesecond plate 172 is secured over the first plate 170, either before orafter casting the gel, to enclose the gel within the channels 176, 178,180, and 182.

With or without the second plate 172 covering the first plate 170,samples of molecules are selectively applied to the channels 176, 178,180, and 182. For example, with the second plate 172 removed from thefirst plate 170, the samples can be applied to the channels by astamping process. As another example, the samples can be applied to thechannels using wicks while the second plate 172 covers the first plate170. Regardless of how the samples are applied, samples can be appliedto all of the channels, or only to selected channels. Each channel canreceive a different sample of molecules, or alternatively more than onechannel can receive the same sample.

Once the samples are applied, the channels 176, 178, 180, and 182provide a plurality of electrophoresis lanes. To selectively generate anelectric field in each lane, a pair of electrodes is associated witheach channel. With reference to the embodiment illustrated in FIG. 5,electrodes 190, 192, 194, and 196 are integrated with the substrate 174at a terminal end of the channels 176, 178, 180, and 182, respectively.Electrodes 200, 202, 204, and 206 are integrated with the substrate 174near a sample-receiving end of the channels 176, 178, 180, and 182,respectively. The electrodes 190, 192, 194, 196, 200, 202, 204, and 206can be formed of a semiconductor material or a conductor material suchas a metal or a polymer.

An electric field is generated in a channel by applying a voltage acrossits associated pair of electrodes. By including a respective pair ofelectrodes for each channel, the electric field in each channel can beindependently controlled.

To sense molecular transport induced by the electric field, each channelcan include a pair of sensing electrodes at a predetermined location asdescribed with reference to the embodiment of FIG. 2. An impedancemeasurement can be performed using each pair of sensing electrodes todetermine the presence of molecules from the sample at the predeterminedlocation.

Alternatively, molecular transport can be sensed by a plurality ofoptical sensors, where at least one optical sensor is associated witheach channel. The plurality of optical sensors can be integrated witheither the first plate 170 or the second plate 172.

The first plate 170 optionally includes a heat sink 208 which functionsin a manner similar to the heat sink 97. The heat sink 208 is located atan opposite face of the substrate 174 relative to the face at which thechannels are defined.

FIG. 6 illustrates a second embodiment of an apparatus for applyingsamples to an electrophoresis device 210. The apparatus includes a stampmember 212 which defines a plurality of transfer elements 214. Thetransfer elements 214 transfer to a plurality of electrophoresis lanes216 samples and/or buffer solutions 218.

The transfer elements 214 are patterned according to locations of theelectrophoresis lanes 216 on the electrophoresis device 210. Using theelectrophoresis device illustrated in FIG. 5, for example, the transferelements 214 can be patterned as a one-dimensional array and spaced inaccordance with the spacing of the electrophoresis channels. Using theelectrophoresis device illustrated in FIG. 2, for example, the transferelements 214 can be patterned as a one-dimensional array and spaced inaccordance with the spacing of the filling regions.

The transfer elements 214 can have any of a variety of forms. In oneform, the transfer elements 214 include a plurality of reservoirsdefined on a surface of the stamp member 212. Each reservoir has theform of a small inclusion in the stamp member 212 to hold a smallquantity of the sample and/or the buffer solution 218. When the stampmember 212 is applied to a surface of the electrophoresis device 210,each sample and/or buffer solution 218 is deposited at its correspondingelectrophoresis lane by a corresponding reservoir.

In a second form, the transfer elements 214 include a plurality ofprojected portions. The plurality of projected portions absorb thesample and/or the buffer solution 218 applied thereto. By contacting thestamp member 212 to the electrophoresis device 210, each sample and/orbuffer solution 218 is transferred to its corresponding electrophoresislane by a corresponding projected portion.

Various embodiments of stamp members and transfer elements, and methodsof using same are described in the copending application "Methods andSystems for Biological Reagent Placement" which is incorporated byreference into the disclosure of the present application.

FIG. 7 illustrates a third embodiment of an apparatus for applyingsamples 230 and a buffer solution 232 to an electrophoresis device 234.The apparatus includes a sample wicking device 236 and a buffer wickingdevice 240. The sample wicking device 236 includes a plurality of wicks242. The wicks 242 are arranged to mate with a plurality ofelectrophoresis lanes 244 at a sample-receiving end of theelectrophoresis device 234. The buffer wicking device 240 includes aplurality of wicks 246. The wicks 246 are arranged to mate with theelectrophoresis lanes 244 at either the sample-receiving end or aterminal end of the electrophoresis device 234.

Using the electrophoresis device illustrated in FIG. 5, for example, thewicks 242 and the wicks 246 can be arranged in a one-dimensional arraywith adjacent wicks being spaced in accordance with the spacing of theelectrophoresis channels. Using the electrophoresis device illustratedin FIG. 2, for example, the wicks 242 and the wicks 246 can be arrangedin a one-dimensional array with adjacent wicks being spaced inaccordance with the spacing of the filling regions.

The wicks 246 transfer the buffer solution 232 absorbed therein to theelectrophoresis lanes 244. Thereafter, the buffer solution 232 diffusesto span the length of the electrophoresis lanes 244.

The wicks 242 transfer the samples 230 absorbed therein to theelectrophoresis lanes 244. Each wick can be utilized to transfer adifferent sample to a different electrophoresis lane of theelectrophoresis device, or can be utilized to transfer a common sampleto a number of electrophoresis lanes.

FIG. 8 illustrates an embodiment of an electrophoresis system inaccordance with the present invention. The electrophoresis systemincludes an electrophoresis device 250 having a plurality ofelectrophoresis lanes. Associated with the plurality of lanes are aplurality of molecular sensors 252 and a plurality of electrodes 254 forgenerating an electric field. Preferably, the electrophoresis device 250is selected from the various embodiments of electrophoresis devicesdescribed herein.

The electrophoresis system further includes a computer system 256 or alike processing apparatus which commands various operations performedusing the electrophoresis device 250. Typically, the computer system 256includes: at least one input device such as a keyboard and/or a pointingdevice such as a mouse; a processor; a memory; at least one storagedevice such as a hard disk drive, a floppy disk drive, and/or an opticalstorage drive; and a display device such as a monitor. The operation ofthe computer system 256 is directed using computer-readable data storedby a computer-readable storage medium, such as a floppy disk, a harddisk, an optical disk, or the memory.

The computer system 256 commands a driver circuit 260 to selectivelyapply and remove a voltage to selected ones of the electrodes 254. Thedriver circuit 260 includes a plurality of electrophoresis drivers, eachcorresponding to one or more electrophoresis lanes, to provide power toproduce predetermined electric field conditions in each of theelectrophoresis lanes. Using this configuration, the electrophoresisprocess can be individually controlled in each of the electrophoresislanes.

The computer system 256 commands a sensing instrument 262 to sense forthe presence of sample molecules using the molecular sensors 252. In apreferred embodiment, each molecular sensor includes a pair ofelectrodes as described with reference to FIG. 2. Here, it is preferredthat the sensing instrument 262 include an impedance meter to measure animpedance of the material between each pair of electrodes. The impedancemeter can perform either an AC (alternating current) impedancemeasurement or a DC (direct current) impedance measurement.

FIG. 9 is a flow chart of an embodiment of a method of electrophoresisin accordance with the present invention. Many of the steps of themethod can be directed by the computer system 256 described withreference to FIG. 8. Although the method is described forelectrophoresis of a single unknown sample using one or moreelectrophoresis lanes, it is noted that the method can becontemporaneously performed for each of a plurality of different unknownsamples in a plurality of electrophoresis lanes.

As indicated by block 270, the method includes a step of applying asample to at least one electrophoresis lane. Typically, the sampleincludes molecules having an unknown characteristic which is to bedetermined using the method.

The sample can be applied to the at least one electrophoresis lane usingthe apparatus described with reference to FIG. 4, FIG. 6, or FIG. 7. Thesample can be applied to at least one electrophoresis channel whichprovides the at least one electrophoresis lane, or to a filling regionassociated with the at least one electrophoresis lane.

Preferably, the at least one electrophoresis lane includes a pluralityof electrophoresis lanes. By applying the same sample to a plurality ofelectrophoresis lanes, a statistical analysis of results generatedthereby can be performed.

As indicated by block 272, the method optionally includes a step ofapplying a known sample to at least one calibration lane. The knownsample typically includes molecules having a known characteristic. Insequencing applications, the known sample can include a plurality ofpredetermined oligonucleotide fragments, or a plurality ofoligonucleotide fragments having known lengths.

The known sample is applied to either at least one electrophoresischannel which provides the at least one calibration lane or to a fillingregion associated with the at least one calibration lane. The knownsample can be applied to the at least one calibration lane using theapparatus described with reference to FIG. 4, FIG. 6, or FIG. 7.

Preferably, the at least one calibration lane includes a plurality ofelectrophoresis lanes. By applying the known sample to a plurality ofelectrophoresis lanes, a statistical analysis of results generatedthereby can be performed.

It is noted that the sample and the known sample can be substantiallysimultaneously applied to their respective lanes, or can be sequentiallyapplied to their respective lanes.

As indicated by block 274, the method includes a step of generating atleast one electric field to electrophorese the sample, and optionally,to electrophorese the known sample. The at least one electric field canbe substantially simultaneously applied to the at least oneelectrophoresis lane and to the at least one calibration lane, or can besequentially applied to the at least one electrophoresis lane and the atleast one calibration lane.

Using the system described with reference to FIG. 8, the step ofgenerating the at least one electric field includes the computer system256 communicating a signal to the driver 260. The signal indicates whichlanes are to have an electric field generated therein. In response toreceiving the signal, the driver 260 generates a predetermined voltagedifference across predetermined pairs of the electrodes 254. In general,the driver 260 can provide either a continuous signal or a pulsed signalfor each pair of the electrodes 254.

As indicated by block 276, the method includes a step of maintaining aquantity indicative of a progression of the electrophoresis. Thequantity, herein referred to as a progression quantity, can indicate atime duration over which an electric field is applied to a lane.Alternatively, the progression quantity can indicate a count of pulsesapplied to an electrode pair by the driver 260. Generally, a respectiveprogression quantity can be maintained for each lane. Each progressionquantity can be maintained, e.g. can be determined and stored, using thecomputer system 256.

As indicated by block 278, the method includes an optional step ofremoving the electric field from the at least one electrophoresis lane,and optionally, the at least one calibration lane. The step of removingthe electric field includes the computer system 256 communicating asignal to the driver 260. The signal indicates which lanes are to haveits electric field removed. In response to receiving the signal, thedriver 260 provides a voltage difference of zero across predeterminedpairs of the electrodes 254.

As indicated by block 280, the method includes a step of sensing for apresence of sample molecules in the at least one electrophoresis lane.As indicated by block 282, the method optionally includes a step ofsensing for a presence of known sample molecules in the at least onecalibration lane.

The steps of sensing can be initiated by the computer system 256communicating a request signal to the sensing instrument 262. Therequest signal indicates which selected ones of the molecular sensors252 are to be utilized by the sensing instrument 262. In response to therequest signal, the sensing instrument 262 can apply and/or receive asignal from each selected one of the molecular sensors 252. Based uponthe applied signal and the received signal, the sensing instrument 262forms a measurement quantity. The sensing instrument 262 communicates asignal representative of the measurement quantity to the computer system256.

As Indicated by block 284, the method includes a step of recording dataassociated with the progression quantity and the results of the sensingsteps. The data is recorded in a memory or a computer-readable storagemedium associated with the computer system 256.

The data can include any one or more of: the measurement quantity, theprogression quantity, a function of the measurement quantity, and afunction of the progression quantity. For example, the data can includea data pair comprising the measurement quantity and the progressionquantity. Alternatively, the data can include the progression quantityif the measurement quantity satisfies a predetermined condition. Variouspredetermined conditions of the measurement quantity are subsequentlydescribed with reference to FIG. 10.

Flow of the method can be directed to repeat the steps indicated byblocks 274, 276, 278, 280, 282, and 284 a number of times. As a result,a collection of data is produced and stored as the sample and the knownsample migrate through the lanes. After forming the collection of data,flow of the method is directed to block 286.

Block 286 indicates a step of determining a characteristic of the samplebased on the collection of data. Preferably, the characteristic is basedupon either the mass of molecules in the sample, the size of moleculesin the sample, or the length of molecule chains in the sample. Ofparticular interest is where the characteristic of the sample is basedupon numbers of bases in DNA fragments or RNA fragments in the sample.

If the sample is electrophoresed in parallel by a plurality ofelectrophoresis lanes, and/or if the known sample is electrophoresed inparallel by a plurality of calibration lanes, the step of determiningthe characteristic can include a step of performing a statisticalanalysis of data generated thereby. The statistical analysis can includecomputing an estimate based on an estimator such as a mean, a trimmedmean, a median, and/or a maximum likelihood estimator for at least asubset of the data. Additionally, the statistical analysis can includecomputing a measure of variability of the subset of the data, such as astandard deviation or a variance. The measure of variability may be usedto compute a confidence level for the estimate.

FIG. 10 illustrates an example plot of a measured quantity versus aprogression quantity. The measured quantity, which can be an impedance,assumes a nominal level L when there is an absence of sample moleculesproximate to a molecular sensor. As sample molecules electrophorese andbecome proximate to the molecular sensor, the measured quantity deviatesfrom the nominal level. Thereafter, as the sample molecules depart fromthe molecular sensor, the measured quantity reverts to the nominallevel.

The example plot is produced by four bands of molecules which cross themolecular sensor during electrophoresis. The four bands cross themolecular sensor at progression quantities of Q1, Q2, Q3, and Q4. Theprogression quantity at which the band crosses the molecular sensor canbe defined by detecting any of the following conditions: (i) a crossingof the measurement quantity beyond a threshold T; (ii) a local extremumpoint 290; (iii) a difference between the measurement quantity and aprevious value of the measurement quantity; or (iv) a rate of change ofthe measurement quantity. The condition can be based on either the localextremum point 290, a leading edge 292, or a trailing edge 294 of theplot of the measurement quantity. Regardless of how the progressionquantities Q1, Q2, Q3, and Q4 are defined, the data recorded in the stepindicated by block 284 preferably includes the values of Q1, Q2, Q3, andQ4. In this manner, a progression quality, such as a time interval for amolecule to be transported to a fixed sensor, is measured. Theprogression quantity, such as the time interval, can be mapped to aphysical property of the molecule, such as the molecular size.

FIG. 11 is a flow chart of an embodiment of a method of determining acharacteristic of the sample based on a collection of data. Variouscharacteristics, including size, mass, or length of molecules in thesample can be determined. For the purpose of illustration, the method isdescribed for the characteristic being a size (i.e. a length or a numberof nucleotide bases) of DNA fragments or RNA fragments within thesample.

As indicated by block 300, the method includes a step of determining atransformation between the progression quantity and the characteristicusing data for the known sample. Assuming the plot of FIG. 10 isgenerated from electrophoresis of the known sample, the data for theknown sample includes the values of Q1, Q2, Q3 and Q4, and known sizesS1, S2, S3, and S4 (in increasing order) of the fragments. Thetransformation can include a linear or a nonlinear curve fit between theknown sizes and the progression quantities.

As indicated by block 302, the method includes a step of transformingprogression quantity data associated with the sample to formcharacteristic data. The progression quantity data is transformed usingthe transformation determined in the previous step.

FIG. 12 illustrates an example of a linear transformation fordetermining sizes of molecules in the sample. The linear transformationis provided by a line 310 fit to a collection of data including the datapairs (Q1, S1), (Q2, S2), (Q3, S3), and (Q4, S4). The lineartransformation can be computed using a line fitting routine such asleast squares regression. Using the linear transformation, a band ofmolecules in the sample detected at a progression quantity value of Qtransforms to a size of S.

FIG. 13 illustrates an example of a second transformation fordetermining sizes of molecules in the sample. The second transformationis provided by a piecewise-linear curve 312 fit to the collection ofdata of (Q1, S1), (Q2, S2), (Q3, S3), and (Q4, S4). Using the secondtransformation, a band of molecules in the sample detected at aprogression quantity value of Q transforms to a size of S. In thisexample, the size S is determined by interpolating between (Q2, S2) and(Q3, S3).

FIG. 14 illustrates an embodiment of an apparatus for sensing samplemolecules for use in embodiments of the electrophoresis device. Theapparatus includes a first molecular sensor comprising a first electrode320 and a second electrode 322 on opposite sides of an electrophoresischannel 324. The molecular sensor further includes a second molecularsensor comprising a third electrode 326 and a fourth electrode 330 onopposite sides of the electrophoresis channel 324. As with the sensingelectrodes described with reference to FIG. 2, the electrodes 320, 322,326, and 330 can be formed of a conductor or a semiconductor material,and can abut or be insulated from an interior of the electrophoresischannel 324.

The apparatus further includes a circuit, such as a bridge circuit 332,which produces a signal based upon a difference between a parametersensed by the first molecular sensor and a parameter sensed by thesecond molecular sensor. Preferably, the bridge circuit 332 produces asignal based upon a difference between an impedance sensed by the firstmolecular sensor and an impedance sensed by the second molecular sensor.The bridge circuit 332 can include a Wheatstone bridge, for example, toproduce a signal based upon the impedance difference. The bridge circuit332 can apply either an AC signal or a DC signal to the electrodes 320,322, 326, and 330 to detect the impedance difference.

The above-described apparatus is advantageous in sensing samplemolecules in the electrophoresis channel 324 based upon a spatial,differential impedance measurement rather than an absolute impedancemeasurement. Consequently, the sample molecules can be detected withless dependence on the absolute impedance of a gel in theelectrophoresis channel 324.

FIG. 15 is a block diagram of an embodiment of an apparatus 340 forapplying a plurality of samples 342 to an electrophoresis device 344.The apparatus 340 defines a plurality of filling ports 346. Each of thefilling ports 346 receives a respective one of the samples 342. Ifdesired, each of the filling ports 346 can receive a different sample.

Preferably, the filling ports 346 are all accessible for receiving thesamples 342 at a face 348 of the apparatus 340. When the apparatus 340 soriented to receive the samples 342, the face 348 can be a top face ofthe apparatus 340.

Once the samples 342 have been dispensed into the filling ports 346, acover 350 is applied to the face 348. The cover 350 seals the samples342 within the apparatus 340, and preferably, seals the samples 342within the filling ports 346. Preferably, the cover 350 is formed of adeformable material such as a flexible plastic, an elastomer, or rubber.As a result, a pressure applied to the filling ports 346 can he variedby depressing the cover 350.

After applying the cover 350 to the face 348, the samples 342 containedin the apparatus 340 can be dispensed into the electrophoresis device344 in a manner described hereinafter, or can be stored for subsequentdispensing. Although the apparatus 340 generally can be stored in anyorientation when containing the samples 342, in some instances it may bedesired to maintain the orientation of the apparatus 340 so that theface 348 is the top face.

The apparatus 340 further includes a plurality of conduits 352 and aplurality of outlets 354. Each of the conduits 352 provides a fluidiccommunication path between a respective one of the filling ports 346 anda respective one of the outlets 354. As a result, the conduits 352provide a plurality of fluidic communication paths to direct the samples342 from the filling ports 346 to the outlets 354. The samples 342 areforced through the conduits 352 by an application of pressure to thefilling ports 346. The pressurization of the filling ports 346 can beaccomplished by depressing the cover 350.

The outlets 354 are arranged to align with filling regions and/orelectrophoresis lanes 356 defined by the electrophoresis device 344.Typically, the outlets 354 are arranged in a one-dimensional pattern. Itdesired, each of the outlets 354 can be shaped as a gasket as describedwith reference to FIG. 4.

During dispensing, the electrophoresis device 344 can be oriented sothat the electrophoresis lanes 356 are substantially parallel to theplane of the face 348, as illustrated. Alternatively, theelectrophoresis device 344 can be oriented so that the electrophoresislanes 356 are transverse to the plane of the face 348.

For ease in applying the samples 342 to the apparatus 340, it ispreferred that the width of each of the filling ports 348 be greaterthan the width of each of the outlets 354. Further, it is preferred thatthe filling ports 348 be arranged to have a greater pitch than theoutlets 354. To accomplish these preferred specifications, the fillingports 346 are arranged in a two-dimensional pattern, such as atwo-dimensional array, at the face 348. The two-dimensional pattern canbe configured in accordance with a predetermined microplate standard,such as standards for 12-well microplates, 40-well microplates, 96-wellmicroplates, 384-well microplates, and 1728-well microplates, forexample.

FIG. 16 is an illustration of another embodiment of an apparatus 360 forapplying a plurality of samples to an electrophoresis device 362. Theapparatus 360 comprises a body 364 which defines a plurality of fillingports. A representative one of the filling ports is indicated byreference numeral 366. The filling ports 366 are arranged as atwo-dimensional array at a top face 368 of the body 364.

The body 364 further defines a slot 370 sized to receive at least aportion of the electrophoresis device 362. An opening to the slot 370 islocated at a second face 372 of the body 364. Preferably, the secondface 372 is oriented transverse to the top face 368 to provide anelbow-type filling apparatus.

The body 364 can be formed of one or more materials, including but notlimited to, plastic, polystyrene, polypropylene, and Nylon. Preferably,the body 364 is rigid or semi-rigid.

The apparatus 360 includes a plurality of conduits, a representative oneindicated by reference numeral 374, in fluidic communication with thefilling ports 366. The conduits 374 have the form of tubes which definea plurality of outlets, a representative one indicated by referencenumeral 376, at their terminal ends. The outlets 376 are positioned tobe accessible within the slot 370.

Each of the outlets 376 can be sized to fit within a filling regionand/or electrophoresis lane of the electrophoresis device 362.Alternatively, each of the outlets 376 can be shaped and sized as agasket described with reference to FIG. 4.

Preferably, each of the filling ports 366 has a volume which is greaterthan an interior volume of a respective one of the conduits 374 coupledthereto. More preferably, each of the filling ports 366 has a volumegreater than or equal to the sum of the interior volume of the conduitand the volume of the filling region of the electrophoresis device 362.Generally, each of the filling ports 366 has a volume greater than orequal to the sum of the interior volume of the conduit and a volumewhich is to be dispensed.

The apparatus 360 further includes a cover 380 which selectively coversand uncovers the top face 368 of the body 364. To facilitate coveringand uncovering of the top face 368, the cover 380 is pivotably-connectedto the body 364 along an edge 382 using a hinge or the like. The cover380 is placed in an uncovered state, as illustrated, for dispensing thesamples into the filling ports 366. In a covered state, the cover 380seals the samples within the apparatus 360 for storage and fordispensing to the electrophoresis device 362.

The cover 380 includes an interior portion 384 having a concave shape,and an edge portion 386 shaped to contact the top face 368 in thecovered state. In general, the interior portion 384 can have otherconcave shapes than that illustrated. Here, for example, the interiorportion 384 can be dome-shaped.

Preferably, the cover 380 is formed of a deformable material such as aflexible plastic, an elastomer, or rubber. The interior portion 384 andthe edge portion 386 can be formed of the same deformable material, ifdesired. Alternatively, the edge portion 386 can be formed of adifferent material than the interior portion 384.

The cover 380 defines an opening 388 which provides a fluidiccommunication path between a first side and a second side. The opening388 equalizes the pressure of a first environment adjacent the firstside and a second environment adjacent the second side of the cover 380.A pressure difference between the first environment and the secondenvironment can be formed by covering the opening 388.

FIG. 17 is an illustration of the embodiment of the apparatus 360 ofFIG. 16 in a covered state. Here, the cover 380 is placed over the topface 368. In the covered state, the edge portion 386 contacts theperiphery of the top face 368 to provide a seal.

The electrophoresis device 362 is shown inserted into the slot 370. Wheninserted, the filling regions of the electrophoresis device 362 matewith the outlets 376 of the apparatus 360.

The samples within the filling ports 366 are dispensed to theelectrophoresis device 362 by covering the opening 388, and applying aforce to the cover 380. The cover 380 deforms in response to applyingthe force, which in turn increases a pressure on the samples in thefilling ports 366. The increase in pressure acts to force the samplesthrough the conduits 374, toward the outlets 376, and into the fillingregions of the electrophoresis device 362. Once the filling regions arefilled, the force to the cover 380 can be removed. Further, if anelastomeric material is used to form the cover 380, the opening 388 canbe uncovered to restore the cover 380 to its original form.

The electrophoresis device 362 can be removed from the apparatus 360after the samples are applied thereto. Thereafter, the apparatus 360 canbe stored for subsequent dispensing of samples in the sameelectrophoresis device 362 or another like electrophoresis device. Afterdispensing the samples one or more times, the apparatus 360 can becleaned and reused, or can be disposed.

FIG. 18 is a top view of a third embodiment of an apparatus 400 fordispensing a plurality of samples to an electrophoresis device 402. Theapparatus 400 includes a plurality of filling ports, a representativeone being indicated by reference numeral 404, which are arranged inaccordance with a predetermined microplate standard. As illustrated, thefilling ports can be arranged in accordance with a 96-well microplatestandard, for example.

The apparatus 400 includes a plurality of conduits (not specificallyillustrated) and a plurality of outlets (not specifically illustrated)to communicate samples from the filling ports to the electrophoresisdevice 402. The electrophoresis device 402 includes at least 96 fillingregions and/or at least 96 electrophoresis lanes to receive the samples.

To receive the samples, the electrophoresis device 402 is inserted intoa side slot 406 of the apparatus 400. The apparatus 400 can include acover (not specifically illustrated) as described earlier to pump thesamples into the electrophoresis device 402.

Although illustrated for dispensing samples in an electrophoresisdevice, it is noted that the apparatus described with reference to FIGS.15-18 can be used to dispense samples in any assay member. Here, theslot 370 and the side slot 406 can be generally referred to asassay-member-receiving slots which are shaped and sized to receive anassay member.

The assay-member-receiving slot can be sized to simultaneously receive aplurality of assay members. The plurality of assay members can bestacked one above another within the assay-member-receiving slot. Aplurality of electrophoresis devices as described herein can be stackedfor placement within the slot 370 or the side slot 406. The apparatus360 can include a second plurality of outlets in fluidic communicationwith the filling ports 366 to dispense samples to a second one of theelectrophoresis devices. The second plurality of outlets are arranged ina one-dimensional pattern substantially parallel to the one-dimensionalpattern of the outlets 376. The second plurality of outlets can belocated above or below the outlets 376, for example. Other pluralitiesof outlets can be included based upon the number of electrophoresisdevices which are to be filled concurrently.

It is further noted that the apparatus described with reference to FIGS.15-18 can dispense other substances, such as gels and buffers, to anassay member.

FIG. 19 is a block diagram of an embodiment of an automatedelectrophoresis system in accordance with the present invention. Theautomated electrophoresis system includes a gel applicator 420 whichapplies a gel to a substrate 422. Preferably, the substrate 422 isprovided by an embodiment of an electrophoresis device described herein.For example, the substrate 422 can be provided by the electrophoresisdevice described with reference to FIG. 1, the first plate 40 describedwith reference to FIG. 2, or the first plate 170 described withreference to FIG. 5. The gel applicator 420 can include a dispenser todispense the gel to a plurality of channels defined by the substrate422, and a blade to distribute the gel along the plurality of channels.Alternatively, the gel applicator 420 can include a plurality oftransfer elements arranged to stamp lanes of the gel onto the substrate422. Here, the substrate 422 can include a substantially flat surfaceonto which a plurality of electrophoresis lanes is stamped.

The automated electrophoresis system further includes a transportmechanism 424 to transport substrates from the gel applicator 420 to abuffer applicator 426. The transport mechanism 424 can include a linearconveyor, a rotary conveyor, or a robotic arm, for example, to transportthe substrates.

As illustrated, the buffer applicator 426 applies a buffer solution to asubstrate 430 having lanes of gel. Preferably, the buffer applicator 426includes an embodiment of the stamp member 212 described with referenceto FIG. 6, or an embodiment of the buffer wicking device 240 describedwith reference to FIG. 7.

The transport mechanism 424 transports a substrate 432 having the geland the buffer solution to a sample applicator 434. The sampleapplicator 434 applies a plurality of samples to electrophoresis lanessupported by the substrate 432. Preferably, the sample applicator 434includes an embodiment of an apparatus for applying samples to anelectrophoresis device described with reference to FIG. 4, FIG. 6, orFIG. 7.

A substrate 436 having the gel, the buffer solution, and the samples istransported by the transport mechanism 424 to a cover plate applicator440. The cover plate applicator 440 applies a cover plate to thesubstrate 436 to cover a face of the substrate 436 exposing the gel, thebuffer solution, and the samples. Examples of the cover plate includethe second plate 42 described with reference to FIG. 2 and the secondplate 172 described with reference to FIG. 5.

The cover plate applicator 440 can include a robotic arm or the like toapply the cover plate to the substrate 436. Alternatively, the coverplate applicator 440 can direct the substrate 436 utilizing a roboticarm or the like to contact the cover plate.

The transport mechanism 424 transports a substrate 442 having the gel,the buffer solution, the samples, and the cover plate to anelectrophoresis station 444. The electrophoresis station 444electrophoreses the samples supported by the substrate 442. Preferably,the electrophoresis station 444 includes an embodiment of anelectrophoresis system described with reference to FIG. 8 and uses anembodiment of a method of electrophoresis described with reference toFIG. 9. The electrophoresis station 444 can further perform a step oflinking the electrophoresis system to electrodes and molecular sensorsof the substrate 442. The electrophoresis station 444 can be linked tothe electrode and the molecular sensors via a wireline connection or viaa wireless connection.

After electrophoresing the samples, the transport mechanism 424transports the substrate 442 to a separator 446. The separator 446separates a cover plate 450 from a substrate 452 having the gel, thebuffer solution, and the samples. The separator 446 can include arobotic arm or the like to perform the separation.

The cover plate 450 is directed back to the cover plate applicator 440for subsequent application to a substrate. The substrate 452 is directedto a cleaning station 454 to remove the gel, the buffer, and the samplestherefrom. A clean substrate 456 having the gel, the buffer, and thesamples removed therefrom by the cleaning station 454 is directed to thetransport mechanism 424 for subsequent use.

A controller 460 directs the operation of the transport mechanism 424,the gel applicator 420, the buffer applicator 426, the sample applicator434, the cover plate applicator 440, and the electrophoresis station444. The controller 460 orchestrates the actions of the above-identifiedcomponents to concurrently process a plurality of substrates, such asthe substrates 422, 430, 432, 436, 442, 452, and 456, in anassembly-line manner. The controller 460 can include a computer system,such as the computer system 256 described with reference to FIG. 8,which produces control signals to control each of the above-identifiedcomponents.

The automated electrophoresis system can be utilized for applicationssuch as sequencing and fragment sizing. In a sequencing application, asample is prepared for performing a restriction analysis. In particular,one or more restriction enzymes are applied to the sample to form aplurality of fragment samples. The fragment samples are applied to atleast one substrate by the sample applicator 434. If the number offragment samples is greater than a number of electrophoresis lanessupported by a single substrate, the sample applicator 434 distributesthe fragment samples to a plurality of substrates. The controller 460compiles results obtained by the electrophoresis station 444 for one ormore substrates to determine a characteristic of the sample, e.g. a basesequence for the sample. The controller 460 provides a digitalrepresentation of the characteristic which can be stored and/ordisplayed.

FIG. 20 illustrates an embodiment of a transport mechanism in accordancewith the present intention. The transport mechanism includes a firstconveyor 470 which transports substrates between at least two stations,namely a first station and a second station, of an automatedelectrophoresis system. Examples of the first station and the secondstation include, but are not limited to, the gel applicator 420, thebuffer applicator 426, the sample applicator 434, and the cover plateapplicator 440 described with reference to FIG. 19. In general, thefirst conveyor 470 can transport substrates to/from each of theabove-listed applicators.

As illustrated, the first conveyor 470 can have the form of a linearconveyor which either contemporaneously or simultaneously transports aplurality of substrates 472 through the automated electrophoresissystem. With reference to FIG. 19, the first conveyor 470 can transportthe substrate 422 to the gel applicator 422, the substrate 430 to thebuffer applicator 426, the substrate 432 to the sample applicator 434,and the substrate 436 to the cover plate applicator 440 eithercontemporaneously or simultaneously.

The transport mechanism further includes a second conveyor 474 whicheither contemporaneously or simultaneously transports a plurality ofsubstrates 476 from the second station to a third station. The secondstation can include any of the above-listed applicators. Preferably, thethird station includes the separator 446 described with reference toFIG. 19.

As illustrated, the second conveyor 474 can have the form of a rotaryconveyor, such as a carousel, which receives a substrate 480 from thefirst conveyor 470 at a first position 482. A cover plate 484 is appliedto the substrate 480 at the first position 482. The plurality ofsubstrates 476 and a plurality of cover plates 486 associated therewithare transported from the first position 482 to a second position 490.

An electrophoresis station, such as the electrophoresis station 444,electrophoreses samples supported by at least one of the substrates 476between the first position 482 and the second position 490. Theelectrophoresis station can perform steps of electrophoresis andsensing/detection as the second conveyor 474 transports the plurality ofsubstrates 476. Preferably, the electrophoresis station simultaneouslyelectrophoreses samples supported by at least two of the plurality ofsubstrates 476 being transported by the second conveyor 474.

At the second position 490, a substrate 492 is separated from its coverplate 494. A separated substrate 496 can be directed to a cleaningstation, such as the cleaning station 454, for re-use. A separated coverplate 498 is transported back to the first position 482 by the secondconveyor 474 for re-use.

FIG. 21 illustrates another approach to contemporaneouslyelectrophoresing samples supported by a plurality of electrophoresisdevices. In this approach, a plurality of electrophoresis devices 500are placed on a carrier 502. The carrier 502 can have a form of a trayor the like having a substantially planar portion to support theelectrophoresis devices 500.

The samples can be applied to the electrophoresis devices 500 prior tobeing placed on the carrier 502. Alternatively, the samples can beapplied while the electrophoresis devices 500 are supported by thecarrier 502. The samples are either contemporaneously or simultaneouslyelectrophoresed while the electrophoresis devices 500 are supported bythe carrier 502. Preferably, steps of sensing and/or detection areperformed during electrophoresis while the electrophoresis devices 500are supported by the carrier 502.

FIG. 22 is a flow chart of an embodiment of a method of electrophoresisfor a single electrophoresis device. Preferably, the method is performedusing the automated electrophoresis system described with reference toeither FIG. 19 or FIG. 23.

As indicated by block 510, the method includes a step of applying a gelto a substrate of the electrophoresis device. The gel can be appliedusing the gel applicator 420. A step of transporting the substrate tothe gel applicator 420 or transporting the gel applicator 420 to thesubstrate can be performed prior to applying the gel.

As indicated by block 512, the method further includes a step ofapplying a buffer to the substrate. The buffer can be applied using thebuffer applicator 426. A step of transporting the substrate to thebuffer applicator 426 or transporting the buffer applicator 426 to thesubstrate can be performed prior to applying the buffer.

As indicated by block 514, the method includes a step of applying atleast one sample to the substrate. The at least one sample can beapplied using the sample applicator 434. A step of transporting thesubstrate to the sample applicator 434 or transporting the sampleapplicator 434 to the substrate can be performed prior to applying theat least one sample. It is noted that the at least one sample caninclude at least one known sample which is applied for calibrationpurposes as described herein.

As indicated by block 516, the method includes a step of applying acover plate to the substrate. The cover plate can be applied using thecover plate applicator 440. A step of transporting the substrate to thecover plate applicator 440 or transporting the cover plate applicator440 to the substrate can be performed prior to applying the cover plate.

As indicated by block 518, the method includes a step ofelectrophoresing at least one sample supported by the substrate. Thestep of electrophoresing can be performed using the electrophoresisstation 444. A step of transporting the substrate to the electrophoresisstation 444 or transporting the electrophoresis station 444 to thesubstrate can be performed prior to performing the step ofelectrophoresis.

Preferably, the step of electrophoresing the at least one sampleincludes a step of sensing a migration of the at least one sample usingat least one molecular sensor associated with either the substrate 442or the cover plate. Further, either the substrate 442 or the cover platecan include a transmitter for wirelessly transmitting a signal derivedfrom the at least one molecular sensor. For example, the transmitter canbe included in a radio frequency tag or a transponder integrated witheither the substrate 442 or the cover plate.

To wirelessly receive the signal from the transmitter, theelectrophoresis station can include a wireless receiver. For example,the receiver can be included in a radio frequency tag communicatingdevice to communicate with a radio frequency tag integrated with eitherthe substrate 442 or the cover plate. The tag communicating device canbe utilized to poll a plurality of radio frequency tags associated witha plurality of electrophoresis devices.

As indicated by block 520, the method includes a step of determining acharacteristic of the at least one sample based on the electrophoresis.The characteristic is determined based upon data generated using the atleast one molecular sensor associated with the substrate 442 and/or thecover plate. The characteristic can be determined using theelectrophoresis station 444 and/or the controller 460.

As indicated by block 522, the method includes a step of removing thecover plate from the substrate. The cover plate can be removed using theseparator 446. A step of transporting the substrate to the separator 446or transporting the separator 446 to the substrate can be performedprior to removing the cover plate.

As indicated by block 524, the method includes a step of transportingthe cover plate for subsequent application to a substrate. This step caninclude transporting the cover plate to the cover plate applicator 440.

As indicated by block 526, the method includes a step of washing thesubstrate. The substrate can be washed by the cleaning station 454 toremove the gel, the buffer, and the electrophoresed samples.

As indicated by block 528, the method includes a step of transportingthe substrate for a subsequent use. Thereafter, flow of the method isdirected back to the step indicated by block 510 so that a subsequentsample can be electrophoresed using the substrate.

It is noted that the above-listed steps can be performed in a differentorder than that illustrated in FIG. 22. For example, the cover plate canbe applied to the substrate prior to applying the at least one sample.Further, some of the above-listed steps can be performed concurrently.For example, the steps of transporting the cover plate and transportingthe substrate can be performed concurrently.

FIG. 23 is a block diagram of a second embodiment of an automatedelectrophoresis system in accordance with the present invention. Thisembodiment differs from the embodiment of FIG. 19 in that a cover plateapplicator 530 applies a cover plate to a substrate 532 prior toreceiving the gel, the buffer, and the sample.

A gel applicator 534 applies a gel to a substrate 536 having a coverplate. The gel applicator 534 can apply the gel at either end of aplurality of channels defined by the substrate. The gel can be drawnthrough the channels by applying a greater pressure at a gel-applicationend in comparison to a pressure at an opposite end. The greater pressurecan be produced using a pump or the like. Alternatively, the gel can bedrawn through the channels by applying a reduced pressure at an oppositeend of the channels. The reduced pressure can be produced using a vacuumor the like applied to the opposite end.

A buffer applicator 538 applies a buffer to a substrate 540 having acover plate and the gel. The buffer applicator 538 applies the buffer ateither end of the channels. The buffer can be drawn through the channelsusing wicking devices as described earlier. Alternatively, the buffercan be drawn through the channels by pressurization.

A sample applicator 542 applies samples to a substrate 544 having acover plate, the gel, and the buffer. The sample applicator 542 appliesthe samples at either end of the channels. Preferably, the samples areapplied to one or more filling regions defined by the substrate 544.

The automated electrophoresis station further includes theelectrophoresis station 444, the separator 446, the cleaning station454, the controller 460, and the transport mechanism 424 described withreference to FIG. 19.

Thus, there has been described herein several embodiments includingpreferred embodiments of an electrophoresis apparatus and method.

Because the various embodiments of the present invention include afilling region having a greater dimension than the electrophoresislanes, they provide a significant improvement in relaxing placementtolerances for applying samples thereto.

Additionally, the various embodiments of the present invention asherein-described provide an electrophoresis apparatus havingelectrophoresis lanes which can be individually loaded with samples,individually powered to electrophorese the samples, and can individuallydetect sample migration therein. Further, a statistical analysis of dataobtained from a plurality of electrophoresis lanes in communication withthe filling region can be performed to improve the confidence of samplemolecule detection.

It will be apparent to those skilled in the art that the disclosedinvention may be modified in numerous ways and may assume manyembodiments other than the preferred form specifically set out anddescribed above.

Accordingly, it is intended by the appended claims to cover allmodifications of the invention which fall within the true spirit andscope of the invention.

What is claimed is:
 1. An electrophoresis apparatus comprising:asubstrate; a first filling region supported by the substrate; a firstplurality of electrophoresis lanes supported by the substrate, the firstplurality of electrophoresis lanes in communication with the firstfilling region; a second filling region supported by the substrate; anda second plurality of electrophoresis lanes supported by the substrate,the second plurality of electrophoresis lanes in communication with thesecond filling region; wherein the first plurality of electrophoresislanes is adjacent the second plurality of electrophoresis lanes; andwherein a distance between the first plurality of electrophoresis lanesand the second plurality of electrophoresis lanes is greater than adistance between each adjacent pair of the first plurality ofelectrophoresis lanes.
 2. The electrophoresis apparatus of claim 1further comprising at least one molecular sensor associated with thefirst plurality of electrophoresis lanes.
 3. The electrophoresisapparatus of claim 2 wherein the at least one molecular sensor isintegrated with the substrate.
 4. The electrophoresis apparatus of claim2 wherein the at least one molecular sensor includes a first electrodeand a second electrode proximate to an electrophoresis lane of the firstplurality of electrophoresis lanes.
 5. The electrophoresis apparatus ofclaim 4 wherein the first electrode and the second electrode are locatedat opposite sides of the electrophoresis lane.
 6. The electrophoresisapparatus of claim 5 further comprising a first insulating layer whichinsulates the first electrode from the electrophoresis lane and a secondinsulating layer which insulates the second electrode from theelectrophoresis lane.
 7. The electrophoresis apparatus of claim 5wherein the first electrode and the second electrode abut with aninterior portion of the electrophoresis lane.
 8. The electrophoresisapparatus of claim 2 wherein the at least one molecular sensor includesa plurality of molecular sensors distributed along the electrophoresislane.
 9. The electrophoresis apparatus of claim 2 wherein the at leastone molecular sensor includes a first molecular sensor and a secondmolecular sensor, the electrophoresis apparatus further comprising abridge circuit which produces a signal based upon a difference between afirst impedance sensed by the first molecular sensor and a secondimpedance sensed by the second molecular sensor.
 10. The electrophoresisapparatus of claim 1 further comprising a plurality of molecularsensors, the plurality of molecular sensors including a respectivemolecular sensor for each of the first plurality of electrophoresislanes.
 11. The electrophoresis apparatus of claim 10 wherein theplurality of molecular sensors is integrated with the substrate.
 12. Theelectrophoresis apparatus of claim 1 wherein the substrate defines aplurality of channels which provide the first plurality ofelectrophoresis lanes.
 13. The electrophoresis apparatus of claim 12wherein the plurality of channels are formed during molding of thesubstrate.
 14. The electrophoresis apparatus of claim 12 wherein theplurality of channels are etched into the substrate.
 15. Theelectrophoresis apparatus of claim 1 further comprising a heat sinkintegrated with the substrate, the heat sink to equalize temperatures ofthe first plurality of electrophoresis lanes with temperatures of thesecond plurality of electrophoresis lanes.
 16. The electrophoresisapparatus of claim 15 wherein the heat sink is integrated with thesubstrate at an opposite face relative to a face at which the firstplurality of electrophoresis lanes is supported.
 17. The electrophoresisapparatus of claim 15 wherein the heat sink includes a thermoelectricmember.
 18. An electrophoresis apparatus comprising:a substrate; aplurality of electrophoresis lanes supported by the substrate; a fillingregion supported by the substrate, the filling region having asample-receiving opening at a first end and openings to the plurality ofelectrophoresis lanes at a second end; wherein a dimension of thefilling region is greater at the sample-receiving opening than at theopenings to the plurality of electrophoresis lanes; and wherein an axisdefined from the first end to the second end of the filling region issubstantially parallel to the plurality of electrophoresis lanes.
 19. Anelectrophoresis apparatus comprising:a substrate; a first filling regiondefined by the substrate, the first filling region having a firstsample-receiving opening at a first sample-receiving end and a firstplurality of openings at a first channel-opening end; a first pluralityof electrophoresis channels defined by the substrate, the firstplurality of electrophoresis channels in communication with the firstplurality of openings of the first filling region, the first pluralityof electrophoresis channels substantially parallel to a first axis fromthe first sample-receiving end to the first channel-opening end of thefirst filling region; a first plurality of molecular sensors including arespective molecular sensor for each of the first plurality ofelectrophoresis channels; a second filling region defined by thesubstrate, the second filling region having a second sample-receivingopening at a second sample-receiving end and a second plurality ofopenings at a second channel-opening end; a second plurality ofelectrophoresis channels defined by the substrate, the second pluralityof electrophoresis channels in communication with the second pluralityof openings of the second filling region, the second plurality ofelectrophoresis channels substantially parallel to a second axis fromthe second sample-receiving end to the second channel-opening end of thefirst filling region; wherein the first plurality of electrophoresischannels is adjacent to the second plurality of electrophoresischannels, and a distance between the first plurality of electrophoresischannels and the second plurality of electrophoresis channels is greaterthan a distance between adjacent pairs of the first plurality ofelectrophoresis channels; and a second plurality of molecular sensorsincluding a respective molecular sensor for each of the second pluralityof electrophoresis channels.
 20. A method of electrophoresis, the methodcomprising the steps of:providing an electrophoresis device having afirst filling region, a second filling region adjacent the first fillingregion, a first plurality of electrophoresis lanes in communication withthe first filling region, and a second plurality of electrophoresislanes in communication with the second filling region and adjacent thefirst plurality of electrophoresis lanes, wherein a distance between thefirst plurality of electrophoresis lanes and the second plurality ofelectrophoresis lanes is greater than a distance between each adjacentpair of the first plurality of electrophoresis lanes and each adjacentpair of the second plurality of electrophoresis lanes; applying a firstsample to the first filling region, the first plurality ofelectrophoresis lanes receiving the first sample from the first fillingregion; applying a second sample to the second filling region, thesecond plurality of electrophoresis lanes receiving the second samplefrom the second filling region; electrophoresing the first sample in thefirst plurality of electrophoresis lanes; and electrophoresing thesecond sample in the second plurality of electrophoresis lanes.
 21. Amethod of electrophoresis, the method comprising the steps of:providingan electrophoresis device having a filling region and a plurality ofelectrophoresis lanes in communication with the filling region, thefilling region having a sample-receiving opening at a first end andopenings to the plurality of electrophoresis lanes at a second end,wherein a dimension of the filling region is greater at thesample-receiving opening than at the openings to the plurality ofelectrophoresis lanes, and wherein an axis defined from the first end tothe second end of the filling region is substantially parallel to theplurality of electrophoresis lanes; applying a sample to the fillingregion, the plurality of electrophoresis lanes receiving the sample fromthe filling region; and electrophoresing the sample in the plurality ofelectrophoresis lanes.